Glycopolysialylation of non-blood coagulation proteins

ABSTRACT

A water soluble polymer, in particular polysialic acid (PSA) or a modified PSA (mPSA), is conjugated to an oxidized carbohydrate moiety of a glycoprotein other than a blood coagulation protein or to a ganglioside or drug delivery system by contacting the oxidized carbohydrate moiety with the water soluble polymer, wherein said water soluble polymer contains an aminooxy group and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the water soluble polymer or wherein said water soluble polymer contains a hydrazide group and a hydrazone linkage is formed between the oxidized carbohydrate moiety and the hydrazide group on the water soluble polymer. Conjugates of aminooxy- or hydrazide-water soluble polymer, such as PSA and mPSA, are thus obtained in which the PSA or mPSA is attached via a carbohydrate moiety.

FIELD OF THE INVENTION

The present invention relates to materials and methods for conjugatingwater soluble polymers, in particular polysialic acid, tocarbohydrate-containing compounds, in particular glycoproteins otherthan a blood coagulation proteins, and to the conjugates obtained.

BACKGROUND OF THE INVENTION

Conjugation of polypeptide drugs such as by PEGylation orpolysialylation protects them from degradation in the blood circulationand thus improves their pharmacodynamic and pharmacokinetic profiles(Harris and Chess, Nat Rev Drug Discov. 2003; 2:214-21). The PEGylationprocess attaches repeating units of ethylene glycol (polyethylene glycol(PEG)) to a polypeptide drug. PEG molecules have a large hydrodynamicvolume (5-10 times the size of globular proteins), are highly watersoluble and hydrated, non-toxic, non-immunogenic and rapidly clearedfrom the body. PEGylation of molecules can lead to increased resistanceof drugs to enzymatic degradation, increased half-life in vivo, reduceddosing frequency, decreased immunogenicity, increased physical andthermal stability, increased solubility, increased liquid stability, andreduced aggregation. The first PEGylated drugs were approved by the FDAin the early 1990s. Since then, the FDA has approved several PEGylateddrugs for oral, injectable, and topical administration.

Sialic acids (also called N-acetyl neuraminic acids) and polysialicacids are found widely distributed in animal tissues and to a lesserextent in other species ranging from plants and fungi to yeasts andbacteria, mostly in glycoproteins and gangliosides.

The abbreviation “PSA” used herein refers to the term “polysialic acid”.Similarly, the term “mPSA” used herein refers to the term “modifiedpolysialic acid”.

PSAs consist of polymers (generally homopolymers) of N-acetylneuraminicacid. The secondary amino group normally bears an acetyl group, but itmay instead bear a glycolyl group. Possible substituents on the hydroxylgroups include acetyl, lactyl, ethyl, sulfate, and phosphate groups.

N-Acetylneuraminic acid Neu5Ac Structure of Sialic Acid(N-Acetylneuraminic Acid)

PSAs and mPSAs generally comprise linear polymers consisting essentiallyof N-acetylneuraminic acid moieties linked by 2,8- or 2,9-glycosidiclinkages or combinations of these (e.g. alternating 2,8- and2,9-linkages). In particularly preferred PSAs and mPSAs, the glycosidiclinkages are α-2,8. Such PSAs and mPSAs are conveniently derived fromcolominic acids, and are referred to herein as “CAs” and “mCAs”. TypicalPSAs and mPSAs comprise at least 2, preferably at least 5, morepreferably at least 10 and most preferably at least 20N-acetylneuraminic acid moieties. Thus, they may comprise from 5 to 500N-acetylneuraminic acid moieties, preferably from 10 to 300N-acetylneuraminic acid moieties. PSAs and CAs can be polymerscomprising different sugar moieties. They can be copolymers. PSAs andCAs preferably are essentially free of sugar moieties other thanN-acetylneuraminic acid. PSAs and CAs preferably comprise at least 90%,more preferably at least 95% and most preferably at least 98%N-acetylneuraminic acid moieties.

Where PSAs and CAs comprise moieties other than N-acetylneuraminic acid(as, for example in mPSAs and mCAs) these are preferably located at oneor both of the ends of the polymer chain. Such “other” moieties may, forexample, be moieties derived from terminal N-acetylneuraminic acidmoieties by oxidation or reduction.

For example, WO-A-0187922 describes such mPSAs and mCAs in which thenon-reducing terminal N-acetylneuraminic acid unit is converted to analdehyde group by reaction with sodium periodate. Additionally, WO2005/016974 describes such mPSAs and mCAs in which the reducing terminalN-acetylneuraminic acid unit is subjected to reduction to reductivelyopen the ring at the reducing terminal N-acetylneuraminic acid unit,whereby a vicinal diol group is formed, followed by oxidation to convertthe vicinal diol group to an aldehyde group.

Sialic acid rich glycoproteins bind selectin in humans and otherorganisms. They play an important role in human influenza infections.For example, sialic acid can hide mannose antigens on the surface ofhost cells or bacteria from mannose-binding lectin. This preventsactivation of complement. Sialic acids also hide the penultimategalactose residue thus preventing rapid clearance of the glycoprotein bythe galactose receptor on the hepatic parenchymal cells.

Structure of Colominic Acid (Homopolymer of N-Acetylneuraminic Acid)

CAs are produced, inter alia, by particular strains of Escherichia colipossessing the K1 antigen. CAs have many physiological functions. Theyare important as a raw material for drugs and cosmetics.

Comparative studies in vivo with polysialylated and unmodifiedasparaginase revealed that polysialylation increased the half-life ofthe enzyme (Fernandes and Gregoriadis, Biochimica Biophysica Acta 1341:26-34, 1997).

The preparation of conjugates by forming a covalent linkage between thewater soluble polymer and the therapeutic protein can be carried out bya variety of chemical methods. One approach for coupling PSA totherapeutic proteins is the conjugation of the polymers via thecarbohydrate moieties of the protein. Vicinal hydroxyl (OH) groups ofcarbohydrates in proteins can be easily oxidized with sodium periodate(NaIO₄) to form active aldehyde groups (Rothfus and Smith, J Biol Chem1963; 238:1402-10; van Lenten and Ashwell, J Biol Chem 1971;246:1889-94). Subsequently the polymer can be coupled to the aldehydegroups of the carbohydrate by use of reagents containing, for example,an active hydrazide group (Wilchek M and Bayer E A, Methods Enzymol1987; 138:429-42). A more recent technology is the use of reagentscontaining aminooxy groups which react with aldehydes to form oximelinkages (WO 96/40662, WO2008/025856).

Additional examples describing conjugation of a PSA to a therapeuticprotein are described in US Publication No. 2009/0076237 which teachesthe oxidation of rFVIII and subsequent coupling to PSA and other watersoluble polymers (e.g. PEG, HES, dextran) using hydrazide chemistry; WO2008/025856 which teaches oxidation of different coagulation factors,e.g. rFIX, FVIII and FVIIa and subsequent coupling to a polymer, e.g.PEG.

Recently, an improved method was described comprising mild periodateoxidation of sialic acids to generate aldehydes followed by reactionwith an aminooxy group containing reagent in the presence of catalyticamounts of aniline (Dirksen A and Dawson P E, Bioconjugate Chem. 2008;19, 2543-8; and Zeng Y et al., Nature Methods 2009; 6:207-9). Theaniline catalysis dramatically accelerates the oxime ligation, allowingthe use of very low concentrations of reagents.

Notwithstanding the methods available of conjugating water solublepolymers to therapeutic proteins, there remains a need to developmaterials and methods for conjugating water soluble polymers tocarbohydrate-containing compounds other than blood coagulation proteinsthat improve the compound's pharmacodynamic and/or pharmacokineticproperties while minimizing the costs associated with the variousreagents.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for conjugating awater soluble polymer to a carbohydrate-containing compound other than ablood coagulation protein that improve the compound's pharmacodynamicand/or pharmacokinetic properties while minimizing the costs associatedwith the various reagents.

In one embodiment of the invention there is provided a method ofconjugating a water soluble polymer to an oxidized carbohydrate moietyof a carbohydrate-containing compound other than a blood coagulationprotein, comprising contacting the oxidized carbohydrate moiety withwater soluble polymer under conditions that allow conjugation, whereinsaid water soluble polymer contains an aminooxy group and an oximelinkage is formed between the oxidized carbohydrate moiety and theaminooxy group on the water soluble polymer, or wherein said watersoluble polymer contains a hydrazide group and a hydrazone linkage isformed between the oxidized carbohydrate moiety and the hydrazide groupon the water soluble polymer. The compound may be (1) a glycoproteinother than a blood coagulation protein, (2) a ganglioside, or (3) a drugdelivery system comprising a carbohydrate group.

The carbohydrate moiety may be oxidized using a sugar-specific oxidizingenzyme or by incubation with a buffer comprising an oxidizing agentselected from sodium periodate (NaIO₄), lead tetraacetate (Pb(OAc)₄) andpotassium perruthenate (KRuO₄).

The carbohydrate moiety may be oxidized at a sialic acid, mannose,galactose or glucose residue.

The water-soluble polymer used in the invention can be, but is notlimited to, polyethylene glycol (PEG), branched PEG, PSA, mPSA, CA, mCA,carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid,chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG) polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(l-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC).

In particular embodiments of the invention illustrated in examplesbelow, the water soluble polymer is PEG or branched PEG.

In further particular embodiments of the invention illustrated inexamples below, the water soluble polymer is polysialic acid (PSA) or amodified PSA (mPSA). The PSA or mPSA may have a molecular weight rangeof 350 Da to 120,000 Da, 500 Da to 100,000 Da, 1000 Da to 80,000 Da,1500 Da to 60,000 Da, 2,000 Da to 45,000 Da or 3,000 Da to 35,000 Da.

The PSA or mPSA may be colominic acid or modified colominic acid.

In another embodiment of the invention, the PSA or mPSA is comprised ofabout 5-500 or 10-300 sialic acid units. In yet another embodiment, theaforementioned method is provided wherein the oxidizing agent is sodiumperiodate (NaIO₄).

The method of the invention may comprise oxidizing the water solublepolymer to form an aldehyde group on a terminal sialic acid unit of thewater soluble polymer, and reacting the oxidized water soluble polymerwith an aminooxy linker.

In yet another embodiment of the invention, the aforementioned method isprovided wherein the water soluble polymer is prepared by reacting anactivated aminooxy linker with oxidized water soluble polymer whereinthe linker is a homobifunctional or heterobifunctional linker. Thehomobifunctional linker can have the general formulaNH₂[OCH₂CH₂]_(n)ONH₂, wherein n=1-11, preferably 1-6. The linker mayspecifically be selected from:

-   -   a 3-oxa-pentane-1,5-dioxyamine linker of the formula:

and

-   -   a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:

PSA or mPSA may be oxidized by incubation with a oxidizing agent to forma terminal aldehyde group at the non-reducing end of the PSA.

The method may comprise oxidizing the water soluble polymer to form analdehyde group on a terminal unit of the water soluble polymer, e.g. aterminal sialic acid unit of the PSA or mPSA, and reacting the oxidizedwater soluble polymer with an aminooxy linker. In still anotherembodiment, an aforementioned method is provided wherein the aminooxylinker is 3-oxa-pentane-1,5-dioxyamine. In a related embodiment, theoxidizing agent is NaIO₄.

In another embodiment of the invention, the aforementioned method isprovided wherein the contacting of the oxidized carbohydrate moiety withthe activated water soluble polymer occurs in a buffer comprising anucleophilic catalyst selected from the group consisting of aniline andaniline derivatives.

In yet another embodiment of the invention, an aforementioned method isprovided further comprising the step of reducing an oxime or hydrazonelinkage in the conjugated protein, for example by incubating theconjugated protein in a buffer comprising a reducing compound selectedfrom the group consisting of sodium cyanoborohydride (NaCNBH₃) andascorbic acid (vitamin C). In a related embodiment the reducing compoundis sodium cyanoborohydride (NaCNBH₃).

In another embodiment of the invention, a conjugated glycoproteinproduced by any aforementioned method is provided. In still anotherembodiment of the invention, a conjugated glycoprotein other than ablood coagulation protein, a ganglioside or a drug delivery systemcomprises (a) the said glycoprotein, ganglioside or drug deliverysystem; and (b) at least one aminooxy water soluble polymer bound to theglycoprotein of (a), wherein said aminooxy water soluble polymer isattached to the glycoprotein, ganglioside or drug delivery system viaone or more carbohydrate moieties. In a still further embodiment of theinvention, a conjugated glycoprotein other than a blood coagulationprotein, a ganglioside or a drug delivery system comprises (a) the saidglycoprotein, ganglioside or drug delivery system; and (b) at least onehydrazide water soluble polymer bound to the glycoprotein of (a),wherein said hydrazide water soluble polymer is attached to theglycoprotein ganglioside or drug delivery system via one or morecarbohydrate moieties.

FIGURES

FIG. 1 shows the synthesis of the water soluble di-aminoxy linkers3-oxa-pentane-1,5-dioxyamine and 3,6,9-trioxa-undecane-1,11-dioxyamine.

FIG. 2 shows the preparation of aminooxy-PSA.

DETAILED DESCRIPTION OF THE INVENTION

The pharmacological and immunological properties ofcarbohydrate-containing compounds, such as glycoproteins other thanblood coagulations proteins can be improved by chemical modification andconjugation with water soluble polymer, in particular PEG or PSA ormPSA. The properties of the resulting conjugates generally stronglydepend on the structure and the size of the polymer. Thus, polymers witha defined and narrow size distribution are usually preferred. PSA andmPSA, used in specific examples, can be purified in such a manner thatresults in a final PSA preparation with a narrow size distribution.

Glycoproteins

As described herein, glycoproteins other than blood coagulation proteinsincluding, but not limited to cytokines such as interleukins, alpha-,beta-, and gamma-interferons, colony stimulating factors includinggranulocyte colony stimulating factors, fibroblast growth factors,platelet derived growth factors, phospholipase-activating protein (PUP),insulin, plant proteins such as lectins and ricins, tumor necrosisfactors and related alleles, soluble forms of tumor necrosis factorreceptors, interleukin receptors and soluble forms of interleukinreceptors, growth factors, tissue growth factors, transforming growthfactors such as TGFαs or TGFβs and epidermal growth factors, hormones,somatomedins, pigmentary hormones, hypothalamic releasing factors,antidiuretic hormones, prolactin, chorionic gonadotropin,follicle-stimulating hormone, thyroid-stimulating hormone, tissueplasminogen activator, and immunoglobulins such as IgG, IgE, IgM, IgA,and IgD, erythropoietin (EPO), blood factors other than bloodcoagulation proteins, galactosidases, α-galactosidases,β-galactosidases, DNAses, fetuin, fragments thereof, and any fusionproteins comprising any of the above mentioned proteins or fragmentsthereof together with therapeutic glycoproteins in general arecontemplated by the invention.

As used herein “biologically active derivative” or “biologically activevariant” includes any derivative or variant of a molecule havingsubstantially the same functional and/or biological properties of saidmolecule, such as binding properties, and/or the same structural basis,such as a peptidic backbone or a basic polymeric unit.

An “analog,” “variant” or “derivative” is a compound substantiallysimilar in structure and having the same biological activity, albeit incertain instances to a differing degree, to a naturally-occurringmolecule. For example, a polypeptide variant refers to a polypeptidesharing substantially similar structure and having the same biologicalactivity as a reference polypeptide. Variants or analogs differ in thecomposition of their amino acid sequences compared to thenaturally-occurring polypeptide from which the analog is derived, basedon one or more mutations involving (i) deletion of one or more aminoacid residues at one or more termini of the polypeptide and/or one ormore internal regions of the naturally-occurring polypeptide sequence(e.g., fragments), (ii) insertion or addition of one or more amino acidsat one or more termini (typically an “addition” or “fusion”) of thepolypeptide and/or one or more internal regions (typically an“insertion”) of the naturally-occurring polypeptide sequence or (iii)substitution of one or more amino acids for other amino acids in thenaturally-occurring polypeptide sequence. By way of example, a“derivative” refers to a polypeptide sharing the same or substantiallysimilar structure as a reference polypeptide that has been modified,e.g., chemically.

Variant or analog polypeptides include insertion variants, wherein oneor more amino acid residues are added to a protein amino acid sequenceof the invention. Insertions may be located at either or both termini ofthe protein, and/or may be positioned within internal regions of theprotein amino acid sequence. Insertion variants, with additionalresidues at either or both termini, include for example, fusion proteinsand proteins including amino acid tags or other amino acid labels. Inone aspect, the protein molecule optionally contains an N-terminal Met,especially when the molecule is expressed recombinantly in a bacterialcell such as E. coli.

In deletion variants, one or more amino acid residues in a protein orpolypeptide as described herein are removed. Deletions can be effectedat one or both termini of the protein or polypeptide, and/or withremoval of one or more residues within the protein amino acid sequence.Deletion variants, therefore, include fragments of a protein orpolypeptide sequence.

In substitution variants, one or more amino acid residues of a proteinor polypeptide are removed and replaced with alternative residues. Inone aspect, the substitutions are conservative in nature andconservative substitutions of this type are well known in the art.Alternatively, the invention embraces substitutions that are alsonon-conservative. Exemplary conservative substitutions are described inLehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York(1975), pp. 71-77] and are set out immediately below.

CONSERVATIVE SUBSTITUTIONS SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T YB. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic)K R H Negatively charged (acidic) D E

Alternatively, exemplary conservative substitutions are set outimmediately below.

CONSERVATIVE SUBSTITUTIONS II EXEMPLARY ORIGINAL RESIDUE SUBSTITUTIONAla (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, ArgAsp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys,Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys(K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro(P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, SerVal (V) Ile, Leu, Met, Phe, Ala

Gangliosides

In embodiments, of the invention, gangliosides are conjugated to watersoluble polymers, e.g. PEG or PSA or mPSA. Gangliosides are known toprovide cells with distinguishing surface markers that can serve incellular recognition and cell-to-cell communication. They are useful astherapeutic agents.

Conjugates of the invention may comprise a ganglioside and a watersoluble polymer, in which the ganglioside comprises a glycosphingolipid(ceramide and oligosaccharide) with one or more sialic acids linked onthe sugar chain. Gangliosides can be classified according to how manysialic acid units are present on the molecule. Examples of gangliosidesare GM1, GM2 and GM3 (monosialo-gangliosides), GD1a, GD1b, GD2 and GD3(disialo-gangliosides), GT1b (trisialo-ganglioside) and GQ1(tetrasialo-ganglioside).

For use in the present invention, preferred gangliosides comprise aceramide linked to glucose, which is linked to a first galactose, whichis linked to N-acetylgalactosamine, which is linked to a secondgalactose. This second galactose can be linked to one sialic acid. Thefirst galactose can be linked to one, two, three or four sialic acids.Sialic acids may be linked either as monomers (one on each of thegalactose molecules), or as oligosialic acids (2-4 sialic acids) to thefirst galactose.

Where administered therapeutic gangliosides need to circulate in theblood for long periods. So that their action on target tissues is moreeffective, gangliosides can be polysialylated, for example, by themethod of the invention.

Drug Delivery Systems

In further embodiments, of the invention, drug delivery systems areconjugated to a water soluble polymer, e.g. PEG or PSA or mPSA. Ingeneral, a drug delivery system (DDS) is any molecular or particulateentity which can control the fate and effect of drugs associated withthe entity. DDSs can be separated into two general types. The first typecomprises macromolecules (MDDSs), for instance antibodies,neoglycoproteins as well as synthetic polymers, such aspoly(hydroxypropylmethacrylamide), polylysine and polymerised alkylcyanoacrylates. The association of drugs with various types ofmacromolecular carriers, including monoclonal antibodies to target thedrug to the desired sites is described for instance by Gregoriadis inNature 265, 407-411 (1977). The second type is particulate DDSs (PDDSs),which comprises for instance nanospheres or microspheres, which comprisebiodegradable materials such as albumin or semibiodegradable materialssuch as dextran and alkylcyanoacrylate polymers, or vesicles formed ofnonionic surfactants or liposomes—for details of which see for exampleGregoriadis in NIPS, 4, 146-151 (1989).

Drugs can either be covalently linked to, or passively entrapped into,the DDS. For instance, PDDS comprising surfactant vesicles or liposomesmay entrap hydrophilic or hydrophobic pharmaceutically active compoundsby being formed of an appropriate combination of layers of surfactant orlipid molecules. Pharmaceutically active compounds are usuallycovalently linked to MDDSs, by a bond which may or may not be lysed inthe body, for instance before or after the active compound performs itsfunction.

Many of the MDDSs have an intrinsic (e.g. antibodies) or acquired (e.g.neoglycoproteins) ability to be recognised by target cells or tissuesthrough receptors on the latter's surface. Typically, such DDSs aretaken up specifically by the target upon injection. Specific uptake is,however, limited with the bulk of the DDSs being taken up by other,irrelevant (to therapy) tissues. The reason for this is that antibodiesand other DDS proteins (regardless of their specificity for the target)must be, like other proteins, catabolised at the end of their biologicallife.

Synthetic polymers used in the macromolecular type MDDSs are forinstance poly(hydroxypropylmethacrylamide) polylysine and polymerisedalkyl cyanoacrylates. These may be catabolised in thereticuloendothelial system (RES) or other tissues by appropriatelysosomal enzymes. It would be desirable to reduce the rate ofcatabolism of such biodegradable macromolecular type DDS by some means,for instance by reducing uptake of the DDS by the RES or other tissues,or by reducing degradation by lysosomal enzymes once taken up by theRES.

Particulate DDSs (PDDSs) are, as a rule, removed from the circulation bythe RES. Because of their propensity for the RES, PDDSs are often usedfor the delivery of drugs to these tissues. It is often desirablehowever, that PDDSs are directed to tissues other than those of the RES.To achieve this goal, one must block or delay RES interception of PDDSs.

DDSs for use in the invention may not initially contain glycons. Anoption is to add or otherwise incorporate a glycon into the DDSstructure. Examples of such cases are liposomes incorporating amannosylated or a galactosylated lipid. These glycoliposomes will targetactives to tissues which express a mannose or galactose receptorrespectively.

Where DDSs need to circulate in the blood for long periods so that e.g.uptake by target tissues is more effective (as with hepatic parenchymalcells), they are advantageously polysialylated by the methods of theinvention.

Administration

In one embodiment a conjugated compound of the present invention may beadministered by injection, such as intravenous, intramuscular, orintraperitoneal injection.

To administer compositions comprising a conjugated compound of thepresent invention to human or test animals, in one aspect, thecompositions comprise one or more pharmaceutically acceptable carriers.The terms “pharmaceutically” or “pharmacologically acceptable” refer tomolecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

As used herein, “effective amount” includes a dose suitable for treatinga mammal having a clinically defined disorder.

The compositions may be administered orally, topically, transdermally,parenterally, by inhalation spray, vaginally, rectally, or byintracranial injection. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. Administration by intravenous,intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,retrobulbar, intrapulmonary injection and or surgical implantation at aparticular site is contemplated as well. Generally, compositions areessentially free of pyrogens, as well as other impurities that could beharmful to the recipient.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage will depend on the type of disease to be treated, as describedabove, the severity and course of the disease, whether drug isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the drug, and thediscretion of the attending physician.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of a conjugated compound or protein asdefined herein. The pharmaceutical composition may further comprise apharmaceutically acceptable carrier, diluent, salt, buffer, orexcipient. The pharmaceutical composition can be used for treatingclinically-defined disorders. The pharmaceutical composition of theinvention may be a solution or a lyophilized product. Solutions of thepharmaceutical composition may be subjected to any suitablelyophilization process.

As an additional aspect, the invention includes kits which comprise acomposition of the invention packaged in a manner which facilitates itsuse for administration to subjects. In one embodiment, such a kitincludes a compound or composition described herein (e.g., a compositioncomprising a conjugated protein), packaged in a container such as asealed bottle or vessel, with a label affixed to the container orincluded in the package that describes use of the compound orcomposition in practicing the method. In one embodiment, the kitcontains a first container having a composition comprising a conjugatedprotein and a second container having a physiologically acceptablereconstitution solution for the composition in the first container. Inone aspect, the compound or composition is packaged in a unit dosageform. The kit may further include a device suitable for administeringthe composition according to a specific route of administration.Preferably, the kit contains a label that describes use of thetherapeutic protein or peptide composition.

In one embodiment, the derivative retains the full functional activityof native therapeutic compounds, and provides an extended half-life invivo, as compared to native therapeutic compounds. In anotherembodiment, the derivative retains at least 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 110, 120, 130, 140, or 150 percent (%) biological activity relativeto native compound.

Sialic Acid and PSA

As used herein, “sialic acid moieties” includes sialic acid monomers orpolymers (“polysaccharides”) which are soluble in an aqueous solution orsuspension and have little or no negative impact, such as side effects,to mammals upon administration of the PSA-protein conjugate in apharmaceutically effective amount. PSA and mPSA are characterized, inone aspect, as having 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, or 500 sialic acid units. In certain aspects,different sialic acid units are combined in a chain.

In one embodiment of the invention, the sialic acid portion of the PSAor mPSA compound is highly hydrophilic, and in another embodiment theentire compound is highly hydrophilic. Hydrophilicity is conferredprimarily by the pendant carboxyl groups of the sialic acid units, aswell as the hydroxyl groups. The saccharide unit may contain otherfunctional groups, such as, amine, hydroxyl or sulphate groups, orcombinations thereof. These groups may be present on naturally-occurringsaccharide compounds, or introduced into derivative polysaccharidecompounds. The PSA and mPSA used in the methods and conjugates of theinvention may be further characterized as described above in theBackground of the Invention.

The naturally occurring polymer PSA is available as a polydispersepreparation showing a broad size distribution (e.g. Sigma C-5762) andhigh polydispersity (PD). Because the polysaccharides are usuallyproduced in bacteria carrying the inherent risk of copurifyingendotoxins, the purification of long sialic acid polymer chains mayraise the probability of increased endotoxin content. Short PSAmolecules with 1-4 sialic acid units can also be synthetically prepared(Kang S H et al., Chem Commun. 2000; 227-8; Ress D K and Linhardt R J,Current Organic Synthesis. 2004; 1:31-46), thus minimizing the risk ofhigh endotoxin levels. However PSA preparations with a narrow sizedistribution and low polydispersity, which are also endotoxin-free, cannow be manufactured. Polysaccharide compounds of particular use for theinvention are, in one aspect, those produced by bacteria. Some of thesenaturally-occurring polysaccharides are known as glycolipids. In oneembodiment, the polysaccharide compounds are substantially free ofterminal galactose units.

In various embodiments, the compound is linked to or associated with thePSA or mPSA compound in stoichiometric amounts (e.g., 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:7, 1:8, 1:9, or 1:10, etc.). In variousembodiments, 1-6, 7-12 or 13-20 PSA and/or mPSA units are linked to thecompound. In still other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more PSA and/or mPSA units arelinked to the compound.

Optionally, the compound is modified to introduce glycosylation sites(i.e., sites other than the native glycosylation sites). Suchmodification may be accomplished using standard molecular biologicaltechniques known in the art. Moreover, the compound, prior toconjugation via one or more carbohydrate moieties, may be glycosylatedin vivo or in vitro.

Aminooxy Linkage

In one embodiment of the invention, the reaction of hydroxylamine orhydroxylamine derivatives with aldehydes (e.g., on a carbohydrate moietyfollowing oxidation by sodium periodate) to form an oxime group isapplied to the preparation of conjugates of compound. For example, aglycoprotein is first oxidized with a oxidizing agent such as sodiumperiodate (NaIO₄) (Rothfus J A et Smith E L., J Biol Chem 1963, 238,1402-10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246,1889-94). The periodate oxidation of e.g. glycoproteins is based on theclassical Malaprade reaction described in 1928, the oxidation of vicinaldiols with periodate to form an active aldehyde group (Malaprade L.,Analytical application, Bull Soc Chim France, 1928, 43, 683-96).Additional examples for such an oxidizing agent are lead tetraacetate(Pb(OAc)₄), manganese acetate (MnO(Ac)₃), cobalt acetate (Co(OAc)₂),thallium acetate (TlOAc), cerium sulfate (Ce(SO₄)₂) (U.S. Pat. No.4,367,309) or potassium perruthenate (KRuO₄) (Marko et al., J Am ChemSoc 1997, 119, 12661-2), By “oxidizing agent” a mild oxidizing compoundwhich is capable of oxidizing vicinal diols in carbohydrates, therebygenerating active aldehyde groups under physiological reactionconditions is meant.

The second step is the coupling of the polymer containing an aminooxygroup to the oxidized carbohydrate moiety to form an oxime linkage. Inone embodiment of the invention, this step can be carried out in thepresence of catalytic amounts of the nucleophilic catalyst aniline oraniline derivatives (Dirksen A et Dawson P E, Bioconjugate Chem. 2008;Zeng Y et al., Nature Methods 2009; 6:207-9). The aniline catalysisdramatically accelerates the oxime ligation allowing the use of very lowconcentrations of the reagents. In another embodiment of the inventionthe oxime linkage is stabilized by reduction with NaCNBH₃ to form analkoxyamine linkage.

In one embodiment of the invention, the reaction steps to conjugate PSAor mPSA to a protein are carried out separately and sequentially (i.e.,starting materials (e.g., protein, polymer, etc), reagents (e.g.,oxidizing agents, aniline, etc) and reaction products (e.g., oxidizedcarbohydrate on a protein, activated aminooxy polymer, etc) areseparated between individual reaction steps).

Additional information on aminooxy technology can be found in thefollowing references, each of which is incorporated in their entireties:EP 1681303A1 (HASylated erythropoietin); WO 2005/014024 (conjugates of apolymer and a protein linked by an oxime linking group); WO96/40662(aminooxy-containing linker compounds and their application inconjugates); WO 2008/025856 (Modified proteins); Peri F et al.,Tetrahedron 1998, 54, 12269-78; Kubler-Kielb J and Pozsgay V., J OrgChem 2005, 70, 6887-90; Lees A et al., Vaccine 2006, 24(6), 716-29; andHeredia K L et al., Macromoecules 2007, 40(14), 4772-9.

Advantages of the invention include high recovery of conjugate, highretention of activity of the conjugated glycoprotein compared tounconjugated protein and high conjugation efficiency.

The invention is now illustrated with reference to the followingexamples. Examples 1-3, 9 and 11-27 illustrate specific embodiments ofthe invention. Examples 4-8 and 10 are included as reference examplesfor their relevance to preparation of corresponding conjugates of theinvention.

EXAMPLES Example 1 Preparation of the homobifunctional linkerNH₂[OCH₂CH₂]₂ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₂ONH₂

(3-oxa-pentane-1,5-dioxyamine) containing two active aminooxy groups wassynthesized according to Boturyn et al. (Tetrahedron 1997; 53:5485-92)in a two step organic reaction employing a modified Gabriel-Synthesis ofprimary amines. In the first step, one molecule of2,2-chlorodiethylether was reacted with two molecules ofEndo-N-hydroxy-5-norbornene-2,3-dicarboximide in dimethylformamide(DMF). The desired homobifunctional product was prepared from theresulting intermediate by hydrazinolysis in ethanol, Except whereotherwise specified, this is referred to as the diaminooxy linker inexamples below.

Example 2 Preparation of the homobifunctional linker NH₂[OCH₂CH₂]₄ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₄ONH₂

(3,6,9-trioxa-undecane-1,11-dioxyamine) containing two active aminooxygroups was synthesized according to Boturyn et al. (Tetrahedron 1997;53:5485-92) in a two step organic reaction employing a modifiedGabriel-Synthesis of primary amines. In the first step one molecule ofBis-(2-(2-chloroethoxy)-ethyl)-ether was reacted with two molecules ofEndo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desiredhomobifunctional product was prepared from the resulting intermediate byhydrazinolysis in ethanol.

Example 3 Preparation of Aminooxy-PSA

500 mg of oxidized PSA (MW=18.8 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 8 ml 50 mM sodium acetate buffer,pH 5.5. Next, 100 mg 3-oxa-pentane-1,5-dioxyamine was added. Aftershaking for 2 hrs at room temperature, 44 mg sodium cyanoborohydride wasadded. After shaking for another 4 hrs at 4° C., the reaction mix wasloaded into a Slide-A-Lyzer (Pierce, Rockford, Ill.) dialysis cassette(3.5 kD membrane, regenerated cellulose) and dialyzed against PBS pH 7.2for 4 days. The product was frozen at −80° C. The preparation of theaminooxy-PSA according to this procedure is illustrated in FIG. 2.

Example 4 Coupling of Aminooxy-PSA to rFIX and Purification of theConjugate

To 12.6 mg rFIX, dissolved in 6.3 ml 50 mM sodium acetate buffer, pH6.0, 289 μl of an aqueous sodium periodate solution (10 mM) was added.The mixture was shaken in the dark for 1 h at 4° C. and quenched for 15min at room temperature by the addition of 6.5 μl 1M glycerol. Lowmolecular weight contaminates were removed byultrafiltration/diafiltration (UF/DF) employing Vivaspin (Sartorius,Goettingen, Germany) concentrators (30 kD membrane, regeneratedcellulose). Next, 43 mg aminooxy-PSA was added to the UF/DF retentateand the mixture was shaken for 18 hrs at 4° C. The excess PSA reagentwas removed by hydrophobic interaction chromatography (HIC). Theconductivity of the cooled reaction mixture was raised to 180 mS/cm andloaded onto a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HICcolumn (1.6×2.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodiumchloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugatewas eluted within 2.4 column volumes (CV) with 50 mM HEPES, 6.7 mMcalcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5 ml/min.The preparation was analytically characterized by measuring totalprotein (BCA) and FIX chromogenic activity. For the PSA-rFIX conjugate aspecific activity of 80.2 IU/mg protein was determined (56.4% incomparison to native rFIX). The results are summarized in Table 1.

TABLE 1 Specific BCA FIX:chrom Specific Activity Activity Item [mg/ml][IU/ml] [IU FIX:Chrom/mg BCA [%] rFIX 8.58 1221 142.3 100 PSA-rFIX 1.1592.2 80.2 56.4

Example 5 Coupling of Aminooxy-PSA to rFIX in the Presence of Aniline asNucleophilic Catalyst

To 3.0 mg rFIX, dissolved in 1.4 ml 50 mM sodium acetate buffer, pH 6.0,14.1 μl of an aqueous sodium periodate solution (10 mM) was added. Themixture was shaken in the dark for 1 h at 4° C. and quenched for 15 minat room temperature by the addition of 1.5 μl M glycerol. Low molecularweight contaminates were removed by means of size exclusionchromatography (SEC) employing PD-10 desalting columns (GE Healthcare,Fairfield, Conn.). 1.2 mg oxidized rFIX, dissolved in 1.33 ml 50 mMsodium acetate buffer, pH 6.0 was mixed with 70 μl of aniline (200 mMaqueous stock solution) and shaken for 45 min at room temperature. Next,4.0 mg aminooxy-PSA was added and the mixture was shaken for 2 hrs atroom temperature and another 16 hrs at 4° C. Samples were drawn after 1h, after 2 hrs and at the end of the reaction after 18 hrs. Next, excessPSA reagent and free rFIX were removed by means of HIC. The conductivityof the cooled reaction mixture was raised to 180 mS/cm and loaded onto a5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HIC column(1.6×2.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was elutedwith a linear gradient to 50 mM HEPES, 6.7 mM calcium chloride, 0.005%Tween 80, pH 7.4 in 20 CV with at a flow rate of 5 ml/min.

Example 6 Coupling of Aminooxy-PSA to rFIX and Reduction with NaCNBH₃

To 10.5 mg rFIX, dissolved in 5.25 ml 50 mM sodium acetate buffer, pH6.0, 530 of an aqueous sodium periodate solution (10 mM) was added. Themixture was shaken in the dark for 1 h at 4° C. and quenched for 15 minat room temperature by the addition of 5.3 μl 1M glycerol. Low molecularweight contaminates were removed by means of UF/DF employing Vivaspin(Sartorius, Goettingen, Germany) concentrators (30 kD membrane,regenerated cellulose). Next, 35.9 mg aminooxy-PSA was added to theUF/DF retentate and the mixture was shaken for 2 hrs at roomtemperature. Then 541 of aqueous sodium cyanoborohydride solution (5M)was added and the reaction was allowed to proceed for another 16 hrs.Then the excess PSA reagent was removed by means of HIC. Theconductivity of the cooled reaction mixture was raised to 180 mS/cm andloaded onto a 5 ml HiTrap Butyl FF HIC (GE Healthcare, Fairfield, Conn.)column (1.6×2.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodiumchloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The conjugatewas eluted within 2.4 CV with 50 mM HEPES, 6.7 mM calcium chloride,0.005% Tween 80, pH 7.4 at a flow rate of 5 ml/min.

Example 7 Coupling of Aminooxy-PSA (Linker: NH₂[OCH₂CH₂]₄ONH₂) to rFIXand Purification of the Conjugate

To 5.6 mg rFIX, dissolved in 2.8 ml 50 mM sodium acetate buffer, pH 6.0,102 μl of an aqueous solution of sodium periodate (10 mM) was added. Themixture was shaken in the dark for 1 h at 4° C. and quenched for 15 minat room temperature by the addition of 2.9 μl of 1M glycerol. Lowmolecular weight contaminates were removed by means of UF/DF employingVivaspin (Sartorius, Goettingen, Germany) concentrators (30 kD membrane,regenerated cellulose). Then 19 mg aminooxy-PSA was added to the UF/DFretentate and the mixture was shaken for 18 hrs at 4° C. The excess PSAreagent was removed by means of HIC. The conductivity of the cooledreaction mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrapButyl FF (GE Healthcare, Fairfield, Conn.) HIC column (1.6×2.5 cm),pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM calciumchloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted within 2.4 CVwith 50 mM HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 at aflow rate of 5 ml/min.

Example 8 Coupling of Aminooxy-PSA to rFVIII

To 11 mg rFVIII, dissolved in 11 ml Hepes buffer pH 6 (50 mM Hepes, 5 mMCaCl₂, 150 mM NaCl, 0.01% Tween) 57 μl 10 mM sodium periodate was added.The mixture was shaken in the dark for 30 min at 4° C. and quenched for30 min at 4° C. by the addition of 107 μl of an aqueous 1M glycerolsolution. Then 19.8 mg aminooxy-PSA (18.8 kD) was added and the mixturewas shaken over night at 4° C. The ionic strength was increased byadding a buffer containing 8M ammonium acetate (8M ammonium acetate, 50mM Hepes, 5 mM CaCl₂, 350 mM NaCl, 0.01% Tween 80, pH 6.9) to get afinal concentration of 2.5M ammonium acetate. Next, the reaction mixturewas loaded on a HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) columnwhich was equilibrated with equilibration buffer (2.5M ammonium acetate,50 mM Hepes, 5 mM CaCl₂, 350 mM NaCl, 0.01% Tween 80, pH 6.9). Theproduct was eluted with elution buffer (50 mM Hepes, 5 mM CaCl₂, 0.01%Tween 80, pH 7.4), and the eluate was concentrated by centrifugalfiltration using Vivaspin (Sartorius, Goettingen, Germany) devices with30,000 MWCO.

Example 9 Preparation of the Homobifunctional Linker NH₂[OCH₂CH₂]₆ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₆ONH₂

(3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine) containing two activeaminooxy groups was synthesized according to Boturyn et al. (Tetrahedron1997; 53:5485-92) in a two step organic reaction employing a modifiedGabriel-Synthesis of primary amines. In the first step one molecule ofhexaethylene glycol dichloride was reacted with two molecules ofEndo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desiredhomobifunctional product was prepared from the resulting intermediate byhydrazinolysis in ethanol.

Example 10 Polysialylation of rFIX Employing a Maleimido/Aminooxy LinkerSystem A. Preparation of the Modification Reagent

An Aminooxy-PSA reagent is prepared by use of a maleimido/aminooxylinker system (Toyokuni et al., Bioconjugate Chem 2003; 14, 1253-9).PSA-SH (20 kD) containing a free terminal SH—group is prepared using atwo step procedure: a) Preparation of PSA-NH₂ by reductive amination ofoxidized PSA with NH₄Cl according to WO05016973A1 and b) introduction ofa sulfhydryl group by reaction of the terminal primary amino group with2-iminothiolane (Traut's reagent/Pierce, Rockford, Ill.) as described inU.S. Pat. No. 7,645,860. PSA-SH is coupled to the maleimido-group of thelinker at pH 7.5 in PBS—buffer using a 10-fold molar excess of thelinker and a PSA-SH concentration of 50 mg/ml. The reaction mixture isincubated for 2 hours under gentle shaking at room temperature. Then theexcess linker reagent is removed and the aminooxy-PSA is bufferexchanged into oxidation buffer (50 mM sodium phosphate, pH 6.0) bydiafiltration. The buffer is exchanged 25 times employing a Pellicon XL5kD regenerated cellulose membrane (Millipore, Billerica, Mass.).

B. Modification of rFIX after Prior Oxidation with NaIO₄

rFIX is oxidized in 50 mM sodium phosphate buffer, pH 6.0 employing 100μM sodium periodate in the buffer. The mixture was shaken in the darkfor 1 h at 4° C. and quenched for 15 min at room temperature by theaddition of glycerol to a final concentration of 5 mM. Low molecularweight contaminates were removed by means of size exclusionchromatography (SEC) employing PD-10 desalting columns (GE Healthcare,Fairfield, Conn.). Oxidized rFIX is then spiked with aniline to obtain afinal concentration of 10 mM and mixed with the aminooxy-PSA reagent toachieve a 5 fold molar excess of PSA. The reaction mixture was incubatedfor 2 hours under gentle shaking in the dark at room temperature.

C. Purification of the Conjugates

The excess of PSA reagent and free rFIX is removed by means of HIC. Theconductivity of the reaction mixture is raised to 180 mS/cm and loadedonto a column filled with 48 ml Butyl-Sepharose FF (GE Healthcare,Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 3 M sodiumchloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with a linear gradient of 60% elution buffer (50mM Hepes, 6.7 mM calcium chloride, pH 7.4) in 40 CV. Finally thePSA-rFIX containing fractions are collected and subjected to UF/DF byuse of a 30 kD membrane made of regenerated cellulose (Millipore). Thepreparation is analytically characterized by measuring total protein(BCA) and FIX chromogenic activity. For the PSA-rFIX conjugates preparedwith both variants a specific activity of >50% in comparison to nativerFIX was determined.

Example 11 Preparation of Aminooxy-PSA Reagent

An aminooxy—PSA reagent was prepared according to Example 3. The finalproduct was diafiltrated against buffer, pH 7.2 (50 mM Hepes) using a 5kD membrane (regenerated cellulose, Millipore), frozen at −80° C. andlyophilized. After lyophilization the reagent was dissolved in theappropriate volume of water and used for preparation of PSA-proteinconjugates via carbohydrate modification.

Example 12 Detailed Synthesis of the Aminooxy-PSA Reagent

3-oxa-pentane-1,5 dioxyamine was synthesized according to Botyryn et al(Tetrahedron 1997; 53:5485-92) in a two step organic synthesis asoutlined in Example 1.

Step 1:

To a solution of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide (59.0 g;1.00 eq) in 700 ml anhydrous N,N-dimethylformamide anhydrous K₂CO₃(45.51 g; 1.00 eq) and 2,2-dichlorodiethylether (15.84 ml; 0.41 eq) wereadded. The reaction mixture was stirred for 22 hours at 50° C. Themixture was evaporated to dryness under reduced pressure. The residuewas suspended in 2 L dichloromethane and extracted two times withsaturated aqueous NaCl-solution (each 1 L). The Dichloromethane layerwas dried over Na₂SO₄ and then evaporated to dryness under reducedpressure and dried in high vacuum to give 64.5 g of3-oxapentane-1,5-dioxy-endo-2′,3′-dicarboxydiimidenorbornene as awhite-yellow solid (intermediate 1).

Step 2:

To a solution of intermediate 1 (64.25 g; 1.00 eq) in 800 ml anhydrousEthanol, 31.0 ml Hydrazine hydrate (4.26 eq) were added. The reactionmixture was then refluxed for 2 hours. The mixture was concentrated tothe half of the starting volume by evaporating the solvent under reducedpressure. The occurring precipitate was filtered off. The remainingethanol layer was evaporated to dryness under reduced pressure. Theresidue containing the crude product 3-oxa-pentane-1,5-dioxyamine wasdried in vacuum to yield 46.3 g. The crude product was further purifiedby column chromatography (Silicagel 60; isocratic elution withDichloromethane/Methanol mixture, 9+1) to yield 11.7 g of the pure finalproduct 3-oxa-pentane-1,5-dioxyamine.

Example 13 Preparation of Aminooxy-PSA Polymer

1.3 g of oxidized colominic acid (23 kDa) was dissolved in 18 ml of 50mM sodium acetate pH 5.5±0.02. 20 fold molar excess of1,11-diamino-3,6,9-trioxaundecane (also referred to as3,6,9-trioxa-undecane-1,11-dioxyamine) was dissolved in minimum amountof 50 mM sodium acetate (pH 5.5±0.02) and was added to the PSA solution.The final colominic acid concentration was 62.5 mg/ml. This reactionmixture was incubated for 2±0.1 hr at 22±1.0° C. on a gentle mixer (22oscillations per minute). After this, 0.65 ml of 160 mg/ml NaCNBH₃solution was added to the above reaction mixture so as to make the finalconcentration of 5.00 mg/ml. This was incubated for 3.0±0.20 hours at4.0±1.0° C. on a shaker (22 oscillations per minute) in a endotoxin freeair tight container with enough headspace for mixing. For thepurification, the sample was diluted with 2 mM triethanolamine, pH8.0±0.02 to make final colominic acid concentration of 20 mg/ml. Thereaction mixture was desalted to remove excess of1,11-diamino-3,6,9-trioxaundecane, NaCNBH₃ and byproducts of thereaction. This was followed by desalting on a Sephadex G25 column using20 mM triethanolamine buffer (pH 8.0±0.02). The pH of the desaltedsample was adjusted to pH 7.8-8.0 and was ultrafiltered/diafiltered with20 mM TEA pH 8.0 once and 2 mM triethanolamine (TEA) pH 8.0 twice. Thesample was freeze dried and stored at −80° C.

Alternatively, purification was done in presence of high salt duringdesalting and ultrafiltration/diafiltration (UF/DF) steps. Anionexchange chromatography in high salt was also used to make highly pureaminooxy-PSA. Different molecular weights of aminooxy-PSA weresynthesized.

Example 14 Coupling of Diaminooxy(3,6,9-trioxa-undecane-1,11-dioxyamine)—PSA to β-Galactosidase

For oxidation of β-Galactosidase (β-Gal), different concentrations ofNaIO₄ (ranging from 0.157 mM to 2 mM) were used. 0.5 mg of β-Gal wasoxidized under acidic pH of 5.75 at 4° C. for 30 minutes in the dark.Oxidation was stopped by adding NaHSO₃ to a final concentration of 5 mM.The conjugation reaction was carried out using the oxidized β-Gal withdiaminooxy PSA polymer (22 kDa). The final concentration of polymer inthe reaction mixture was 1.25 mM whereas the concentration of β-Galranged from 0.125 mg/ml to 0.76 mg/ml. All the reactions were done atpH5.75. Sodium cyanoborohydride was added to the reaction mixture to aconcentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4°C. and samples were collected at time intervals of 1, 2 and 24 hours.Conjugates were characterized using SDS PAGE and western blotting. Ashift in the band was seen for the conjugate in SDS PAGE and this wasalso confirmed by western blotting.

Based on the best reactions conditions, 1.9 mg of β-Gal was oxidizedwith 1.5 mM of NaIO₄ for 30 minutes at 4° C. and then oxidation wasstopped by adding NaHSO₃ to a final concentration of 5 mM. Theconjugation reaction was carried out using the oxidized β-Gal withdiaminooxy PSA polymer. The final concentrations of polymer and proteinin the reaction mixture were 1.25 mM and 0.76 mg/ml respectively. Thefinal pH of the reaction mixture was around 5.75. Sodiumcyanoborohydride was added to the reaction mixture to a concentration of50 mM or 3.17 mg/ml. The reaction was carried out at 4° C. for 2 hours.Purified and unpurified conjugates were characterized using SDS PAGE andwestern blotting. A shift in the band was seen for the conjugate in SDSPAGE and this was confirmed by western blotting using anti-PSA antibody.The in vitro activity PSA-βGal conjugates were comparable to nativeprotein using All in one βGal assay kit (Pierce). Further, the overallprocess was scaled up to 3 fold.

Example 15 Coupling of Diaminooxy-PSA to Fetuin

Fetuin and was oxidized with 10 mM NaIO₄ for 60 minutes at 4° C. in thedark and the oxidation was stopped by adding NaHSO₃ to a finalconcentration of 10 mM. The conjugation reaction was carried out usingthe oxidized Fetuin with diaminooxy PSA polymer (23 kDa). The finalconcentration of polymer in the reaction mixture was 2.5 mM at pH 5.75.Sodium cyanoborohydride was added to the reaction mixture to aconcentration of 50 mM or 3.17 mg/ml. The final protein concentration inthe reaction was 0.714 mg/ml and the reaction was carried out at 4° C.for 2 hours. These conjugates were characterized using SDS PAGE andwestern blotting. A shift in the band was seen for the conjugates in SDSPAGE and this was also confirmed by western blotting.

For a scale up reaction, 5 mg Fetuin was oxidized with 10 mM NaIO₄ for60 minutes at 4° C. in dark and then oxidation was stopped by addingNaHSO₃ to a final concentration of 10 mM. The conjugation reaction wascarried out using the oxidized Fetuin with diaminooxy PSA polymer (23kDa). The final concentration of polymer in the reaction mixture was 2.5mM at pH of 5.75. Sodium cyanoborohydride was added to the reactionmixture to a concentration of 50 mM or 3.17 mg/ml. The reaction wascarried out at 4° C. and sample was collected after 2 hours. Purifiedand unpurified conjugates were characterized using SDS PAGE and westernblotting. A shift in the band was seen for the conjugate in SDS PAGE andthis was also confirmed by western blotting.

Example 16 Coupling of Diaminooxy-PSA to Fetuin with Aniline to Act as aNucleophilic Catalyst

0.2 mg of Fetuin was oxidized with 10 mM NaIO₄ for 30 minutes at 4° C.in dark and then oxidation was stopped by adding NaHSO₃ to a finalconcentration of 5 mM. The conjugation reaction was carried out usingthe oxidized Fetuin with diaminooxy PSA polymer (23 kDa). The finalconcentration of polymer in the reaction mixture was 1.25 mM. The finalpH of reaction mixture was 5.75. Sodium cyanoborohydride was added tothe reaction mixture to a concentration of 50 mM or 3.17 mg/ml. Thefinal protein concentration in the reaction was 0.125 mg/ml. 84.21 μl of200 mM aniline solution was added to the 1.6 ml of reaction mixture. Thereaction was carried out at 4° C. overnight.

Example 17 Coupling of Diaminooxy-PSA to Erythropoietin (EPO)

0.2 mg of EPO was oxidized with 10 mM of NaIO₄ for 30 minutes at 4° C.The oxidation was stopped by adding NaHSO₃ to a final concentration of 5mM. The conjugation reaction was carried out using the oxidized EPO withdiaminooxy polymer of 23 kDa. The final concentration of polymer in thereaction mixture was 1.25 mM. The final concentration of EPO in thereaction mixture was 0.125 mg/ml. The final pH of the reaction mixturewas around 5.75. Sodium cyanoborohydride was added to the reactionmixture to a concentration of 50 mM or 3.17 mg/ml. The reaction wascarried out at 4° C. for 24 hours. Unpurified conjugate wascharacterized using SDS PAGE. A shift in the band was seen for theconjugate in SDS PAGE.

Example 18 Coupling of Diaminooxy-PSA to EPO with Aniline to Act as aNucleophilic Catalyst

0.2 mg of EPO was oxidized with 10 mM NaIO₄ for 30 minutes at 4° C. Theoxidation was stopped by adding NaHSO₃ to a final concentration of 5 mM.The conjugation reaction was carried out using the oxidized EPO withdiaminooxy PSA polymer (22 kDa). The final concentration of polymer inthe reaction mixture was 1.25 mM. The final pH of the reaction mixturewas around 5.75. Sodium cyanoborohydride was added to the reactionmixture to a concentration of 50 mM or 3.17 mg/ml. The final proteinconcentration in the reaction was 0.125 mg/ml. 84.21 μl of 200 mManiline solution was added to the 1.6 ml of reaction mixture. Thereaction was carried out at 4° C. for overnight. The conjugates werecharacterized using SDS PAGE. A shift in the band was seen in theconjugates. No adverse effect of aniline was observed on activity of theconjugates.

Example 19 Coupling of Diaminooxy-PSA to DNAse

For glycopolysialylation of DNAse, bovine pancreas DNAse was used forconjugation reaction. This source of DNAse was supplied as lyophilizedpowder, which was stored at −20° C. Prior to the reaction, thislyophilized powder was dissolved in sodium acetate buffer (pH 5.75). Thepolymer used for glycopolysialylation had a weight in the range of 10kDa to 22 kDa. For oxidation of glycon moiety of DNAse, NaIO₄ was usedas oxidizing agent to a final concentration of 1 mM. DNAse was oxidizedat acidic pH of 5.75 at 4° C. for 30 minutes. The oxidation was stoppedby adding NaHSO₃ to a final concentration of 2 mM. After oxidation wascomplete, the conjugation reaction was carried out by addition ofdiaminooxy PSA polymer to a final concentration of 1.25 mM. NaCNBH₃ wasadded to the reaction mixture to a final concentration of 50 mM or 3.17mg/ml and the polysialylation of the DNAse was preformed 4.0±1.0° C. forat least 2 hours. The reactions were stopped with 25 molar excess ofTris over polymer. The conjugates were characterized using SDS PAGE andwestern blotting. A shift in the band was seen for the conjugate in SDSPAGE and positing result was obtained from western blotting. Activitywas measured as 95% (compared with the less than 50% observed incomparable conjugates made using aldehyde linker chemistry).

Example 20 Coupling of Diaminooxy (3 Oxa-Pentane-1,5-DioxyamineLinker)-PSA to β-Galactosidase

For oxidation of β-Galactosidase, NaIO₄ was used at a concentration of 2mM. 3 mg of β-Galactosidase was oxidized at acidic pH of 5.75 at 4° C.for 30 minutes then oxidation was stopped by adding NaHSO₃ to a finalconcentration of 2 mM. The conjugation reaction was carried out usingthe oxidized β-Galactosidase with diaminooxy PSA polymer (23 kDa). Thefinal concentration of polymer in the reaction mixture was 1.5 mM. Thefinal concentration of β-Galactosidase in reaction mixture was 0.867mg/ml. The final pH of reaction mixture was around 5.75. Sodiumcyanoborohydride was added to the reaction mixture to a concentration of50 mM or 3.17 mg/ml. The reaction was carried out at 4° C. for 2 hours.Conjugates were characterized using SDS PAGE and western blotting. Ashift in the band was seen for the conjugate in SDS PAGE and positingresult was obtained from western blotting.

Example 21 Preparation of Hydrazine-Colominic Acid

We used the following protocol to prepare a PSA-hydrazide (colominicacid-hydrazide) using adipic acid dihydrazide. Analogous methods areused to make other PSA-hydrazides.

1 Dissolve 1 g of activated colominic acid in ˜10 ml of 20 mM sodiumacetate pH 5.5±0.02. Final colominic acid concentration should be 62.5mg/ml2 Dissolve 25-fold molar excess (over oxidized colominic acid “CAO”) ofadipic acid dihydrazide (MW=174.2 gms) in minimum amount of 20 mM sodiumacetate (pH 5.5±0.02) and add to solution from 1.

$\begin{matrix}{{{Amount}\mspace{14mu} {of}\mspace{14mu} {adipic}\mspace{14mu} {acid}\mspace{14mu} {dihydrazide}\mspace{14mu} {to}\mspace{14mu} {be}\mspace{14mu} {added}} = \frac{{Weight}\mspace{14mu} {of}\mspace{14mu} {CAO}\mspace{14mu} {in}\mspace{14mu} {grams} \times 25 \times {MW}\mspace{14mu} {of}\mspace{14mu} {adipic}\mspace{14mu} {acid}\mspace{14mu} {dihydrazide}\mspace{14mu} {in}\mspace{14mu} {gms}}{{MW}\mspace{14mu} {of}\mspace{14mu} {CAO}\mspace{14mu} {in}\mspace{14mu} {Daltons}}} \\{= \frac{1 \times 25 \times 174.2}{15 \times 10^{3}}} \\{= {0.290\mspace{14mu} g}}\end{matrix}$

4 After adding adipic acid dihydrazide solution, make up the volume ofcolominic acid with sodium acetate to a final concentration of 62.5mg/ml. Therefore total reaction volume is 16 ml.5 Incubate the reaction mixture for 2±10.1 hr at 22.0±1.0° C. on shaker(22 oscillations per minute).6 Prepare concentrated NaCNBH₃ solution (165 mg/ml) and add 0.5 ml tosolution from 1 so that the final concentration of this becomes 5.0mg/ml in the final reaction mixture. Incubate the reaction mixture for3.0±10.20 hours at 4.0±1.0° C. on shaker (22 oscillations per minute).7 Keep the reaction mixture in endotoxin-free, air tight container withexcess 50 ml of headspace for proper mixing (there should be enoughspace so that reaction mixture should not touch the cap of container).8 After 3 hours reaction at 4° C., dilute the sample with 2 mMtriethanolamine (make the volume up to 50 ml), at pH 8.0±0.02 to makefinal colominic acid concentration to 20 mg/ml.9 Desalt the reaction mixture to remove excess of untreated adipic aciddihydrazide, NaCNBH₃ etc from the polymer. This can be done by GPC(using XK 50 Sephadex G-25 medium matrix; ≤1.8 mg of CA/ml matrix; 35 cmbed height; Column volume=687 ml) by observing UV 224 nm andconductivity. Desalting is carried out with 20 mM triethanolamine (pH8.0±0.02) buffer.10 After desalting, colominic acid-hydrazide is subjected to 1 cycle ofultrafiltration, 1 cycle of diafiltration using 20 mM TEA, pH 8.0±0.02and at least 3 cycles of diafiltration using 2 mM TEA, pH 8.0±0.02. Thiscan be done using 3 kDa vivaflow cassettes.11 Adjust the pH of desalted sample to pH 7.8-8.0. Optionally,freeze-dry the sample and consecutively keep it for secondary drying toremove excess of moisture.

Example 22 Coupling of Hydrazide-PSA to Erythropoietin

For oxidation of erythropoietin (EPO), NaIO₄ was used at a concentrationof 10 mM. EPO (1 mg) was oxidized at pH 5.75 at 4° C. for 30 minutesthen oxidation was stopped by adding NaHSO₃ to a final concentration of5 mM. The conjugation reaction was carried out using the oxidized EPOwith hydrazide-PSA polymer. The molecular weight of the hydrazide-PSAused for conjugation was 24.34 kDa. The final concentration ofhydrazide-PSA in the reaction mixture was 1.25 mM. The finalconcentration of EPO in the reaction mixture was 0.125 mg/ml. The finalpH of the reaction mixture was around 5.75. Sodium cyanoborohydride wasadded to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml.The reaction was carried out at 4° C. for 24 hours. Conjugates werecharacterized using SDS PAGE and western blotting. A shift in the bandwas seen for the conjugate in SDS PAGE and a positive result wasobtained from western blotting.

Example 23 Coupling of Hydrazide-PSA to β-Galactosidase

β-Galactosidase (0.5 to 4.5 mg) was oxidized with 0.625 to 2 mM of NaIO₄for 30 minutes at 4° C. The oxidation was stopped by adding NaHSO₃ to afinal concentration of 5 mM. The conjugation reaction was carried outusing the oxidized β-galactosidase with hydrazide-PSA ranging from 24.34kDa to 27.9 kDa. The final concentration of hydrazide-PSA in thereaction mixture was 1.25 mM. The final concentration of β-galactosidasein the reaction mixture was in a range from 0.125 mg/ml to 0.76 mg/ml.The final pH of reaction mixture should be around 5.75. Sodiumcyanoborohydride was added to the reaction mixture to a concentration of50 mM or 3.17 mg/ml. The reaction was carried out at 4° C. and sampleswere collected at 1, 2 and 24 hours. Purified and unpurified conjugatewas characterized using SDS PAGE and western blotting. A shift in theband was seen for the conjugate in SDS PAGE and a positive result wasobtained from western blotting. Activity was measured as 84%.

Example 24 Coupling of Hydrazide-PSA to Fetuin

Fetuin (0.25 mg) was oxidized with NaIO4 (5 or 10 mM) for 30 or 60minutes at 4° C. The oxidation was stopped by adding NaHSO₃ to a finalconcentration of 5 or 10 mM as appropriate to match the concentration ofNaIO₄ used for oxidation. The conjugation reactions were carried outusing the oxidized Fetuin with adipic acid dihydrazide-PSA polymer. Thefinal concentration of the polymer in the reaction mixture was between1.25 and 2.5 mM. The final pH of reaction mixture was around 5.75.Sodium cyanoborohydride was added to the reaction mixture to aconcentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4°C. for 1 hour to 4 hours. The conjugates were characterized using SDSPAGE and western blotting. A shift in the band was seen for theconjugate in SD S PAGE for each set of reaction conditions and apositive result was obtained from western blotting.

A scaled-up reaction for 5 mg Fetuin followed by purification of theresulting conjugate was carried out. 5 mg Fetuin was oxidized with 10 mMNaIO₄ for 60 minutes at 4° C. and then oxidation was stopped by addingNaHSO₃ to a final concentration of 10 mM. The conjugation reaction wascarried out using the oxidized Fetuin with adipic acid dihydrazide-PSApolymer. The final concentration of polymer in the reaction mixture was2.5 mM. The final pH of reaction mixture was around 5.75. Sodiumcyanoborohydride was added to the reaction mixture to a concentration of50 mM or 3.17 mg/ml. The reaction was carried out at 4° C. and sampleswere collected at 2 hours. Purified and unpurified conjugate wascharacterized using SDS PAGE and western blotting. A shift in the bandwas seen for the conjugate in SDS PAGE and a positive result wasobtained from western blotting.

Example 25 Coupling of Hydrazide-PSA to DNAse

DNAse was oxidized with NaIO4 to a final concentration ranging from 0.2mM to 2 mM for 30 minutes at 4° C. The oxidation reaction was stopped byadding NaHSO₃ to a final concentration of between 2 and 5 mM dependingupon the concentration of NaIO₄ used for oxidation. Glycopolysialylationof oxidized DNAse was carried out by addition of hydrazide-PSA polymerto a final concentration of 1.25 mM to the oxidized DNAse. Sodiumcyanoborohydride was added to the reaction mixture to a finalconcentration of 50 mM or 3.17 mg/ml and the glycopolysialylation of theDNAse was performed at 4° C. for a time period ranging from 1 hour to 2hours. The reactions were stopped with 25-fold molar excess of Tris overpolymer. The conjugates were characterized using SDS PAGE and westernblotting. A shift in the band was seen for the conjugates in SDS PAGEand a positive result was obtained from western blotting. The activitywas measured as 49%.

Example 26 PEGylation of β-Galactosidase Using Aminooxy Linker(3-Oxa-Pentane-1,5-Dioxyamine)

β-Galactosidase (1 mg) was oxidized with 1.5 mM of NaIO₄ for 30 minutesat 4° C. The oxidation was stopped by adding NaHSO₃ to a finalconcentration of 1.5 mM. The conjugation reaction was carried out usingthe oxidized β-Galactosidase with diaminooxy-PEG polymer (20 kDa). Thefinal concentration of the polymer in the reaction mixture was 1.25 mM.The final concentration of β-Galactosidase in the reaction mixture was 1mg/ml. The final pH of reaction mixture should be around 5.75. Sodiumcyanoborohydride was added to the reaction mixture to a concentration of50 mM or 3.17 mg/ml. The reaction was carried out at 4° C. for 2 hours.Unpurified conjugate was characterized using SDS PAGE and a shift in theband was seen for the conjugate in SDS PAGE. The activity was measuredas 59%.

Example 27 PEGylation of Erythropoietin Using Aminooxy Linker

Erythropoietin (EPO; 0.2 mg) was oxidised with 5 or 10 mM NaIO₄ in 50 mMsodium acetate at pH 5.75 for 45 minutes at 4° C. and then oxidation wasstopped by adding NaHSO₃ to a final concentration of 5 or 10 mM (tomatch the concentration of NaIO₄ used for oxidisation). The conjugationreaction was carried out using the oxidised EPO with diaminooxy PEGpolymer (20 kDa). The final concentration of the polymer in the reactionmixture was 1.5 mM. The final pH of reaction mixture should be around5.75. Sodium cyanoborohydride was added to the reaction mixture to aconcentration of 50 mM or 3.17 mg/ml. The final protein concentration inthe reaction was 0.4 mg/ml. The conjugation reaction was carried outovernight at 4° C.

The invention thus provides conjugates of compounds other than bloodcoagulation proteins with water soluble polymers, in particular PSA andmPSA.

1-28. (canceled)
 29. A method of conjugating a polysialic acid (PSA) ora modified PSA (mPSA) to an oxidized carbohydrate moiety of aglycoprotein other than a blood coagulation protein, comprising acarbohydrate group, comprising contacting the oxidized carbohydratemoiety with PSA or mPSA under conditions that allow conjugation, therebyforming a conjugated glycoprotein, wherein said PSA or mPSA contains anaminooxy group, and an oxime linkage is formed between the oxidizedcarbohydrate moiety and the aminooxy group on the PSA or mPSA.
 30. Themethod of claim 29, wherein said conjugated glycoprotein has abiological activity of at least 80% relative to a native glycoprotein.31. The method of claim 29, wherein PSA or mPSA is mPSA, and said mPSAis PSA comprising a moiety derived from a terminal N-acetylneuraminicacid moiety by oxidation or reduction.
 32. The method of claim 29,wherein PSA or mPSA is colominic acid or modified colominic acid. 33.The method of claim 29, wherein PSA or mPSA comprises 2-500 sialic acidunits.
 34. The method of claim 29, wherein the glycoprotein is selectedfrom the group consisting of cytokines such as interleukins, alpha-,beta-, and gamma-interferons, colony stimulating factors includinggranulocyte colony stimulating factors, fibroblast growth factors,platelet-derived growth factors, phospholipase-activating protein (PUP),insulin, plant proteins such as lectins and ricins, tumor necrosisfactors and related alleles, soluble forms of tumor necrosis factorreceptors, interleukin receptors and soluble forms of interleukinreceptors, growth factors, tissue growth factors, transforming growthfactors such as TGFα or TGFβ and epidermal growth factors, hormones,somatomedins, pigmentary hormones, hypothalamic releasing factors,antidiuretic hormones, prolactin, chorionic gonadotropin,follicle-stimulating hormone, thyroid-stimulating hormone, tissueplasminogen activator, immunoglobulins such as IgG, IgE, IgM, IgA, andIgD, monoclonal antibodies, erythropoietin (EPO), blood factors otherthan blood coagulation proteins, galactosidases, α-galactosidases,β-galactosidases, DNAses, fetuin and fragments thereof, and fusionproteins comprising any of the above mentioned proteins or fragmentsthereof.
 35. The method of claim 29, wherein the glycoprotein isselected from the group consisting of tumour necrosis factors andrelated alleles, soluble forms of tumor necrosis factor receptors,immunoglobulins such as IgG, IgE, IgM, IgA and IgD, monoclonalantibodies, erythropoietin (EPO), DNAses, fetuin, fragments thereof andfusion proteins comprising any of the above mentioned proteins orfragments thereof.
 36. The method of claim 29, comprising oxidizing thecarbohydrate moiety by incubating the glycoprotein with sodium periodate(NaIO₄).
 37. The method of claim 29, comprising oxidizing the PSA ormPSA to form an aldehyde group on a terminal unit of the PSA or mPSA,and reacting the oxidized PSA or mPSA with an aminooxy linker.
 38. Themethod of claim 37, comprising oxidizing the PSA or mPSA using NaIO₄.39. The method of claim 29, comprising contacting the oxidizedcarbohydrate moiety with the PSA or mPSA in a buffer comprising anucleophilic catalyst selected from aniline or aniline derivatives. 40.The method of claim 29, wherein said PSA or mPSA contains an aminooxygroup and an oxime linkage is formed between the oxidized carbohydratemoiety and the aminooxy group on the PSA or mPSA.
 41. The method ofclaim 40, wherein the aminooxy group is formed by reacting oxidized PSAor mPSA with an aminooxy linker, and the aminooxy linker is3-oxa-pentane-1,5-dioxyamine or 3,6,9-trioxa-undecane-1,11-dioxyamine.42. The method of claim 29, further comprising reducing an oxime linkagein the conjugated glycoprotein by incubation in the presence of areducing compound.
 43. The method of claim 42, wherein the reducingcompound is sodium cyanoborohydride (NaCNBH₃) or ascorbic acid (vitaminC).
 44. A conjugated glycoprotein, obtained by the method of claim 29.45. The conjugated glycoprotein other than a blood coagulation protein,comprising: (a) the glycoprotein and (b) at least one aminooxy-PSA or-mPSA bound to the glycoprotein of (a), wherein said aminooxy-PSA or-mPSA is attached to the glycoprotein via one or more carbohydratemoieties.